There is now compelling evidence that the pulmonary collectins, surfactant proteins A and D, play a front line role in the innate defense against inhaled pathogens. As pattern recognition molecules, SP-A and SP-D recognize and bind to a large and diverse pool of microorganisms. Early recognition represents the initial steps to mounting an effective immunological response to pulmonary challenge from inhaled microorganisms, allergens, and environmental substances such as pollutants. Failure or delay in such recognition promotes the development of infection, inflammation or acute lung injury. The lung collectins recognize a wide range of major respiratory pathogens, including gram-negative bacteria, gram-positive bacteria, viruses, mycobacteria, mycoplasma, fungi, and parasites. Recent studies demonstrate that the lung collectins directly neutralize certain bacteria and fungi through membrane permeabilization and also influenza A virus (lAV). Yet despite the importance to pulmonary innate defense, these molecular mechanisms remain poorly understood. Specifically, mechanisms through which these innate defense proteins specifically recognize lAV mannans and gram-negative lipopolysaccharide (endotoxin) will be answered through a multi-pronged, highly collaborative, and often iterative approach using complementary techniques: (1) x-ray crystallography, which provides structural information on proteins and protein-ligand complexes at the level of individual atoms; (2) spectroscopy, which extends the crystallographic data to membrane surface or solution states; (3) site-directed mutagenesis coupled with structure-function assays to generate and test mechanistic hypotheses (4) molecular dynamic simulations to extend composite models to dynamic, physiological contexts. Proposed studies focus on trimeric neck-carbohydrate recognition domains (NCRD) as they demonstrate ligand recognition properties and are amenable to high-resolution structural analysis. We have established an extensive crystallographic program focusing on recombinant SP-A and SP-D NCRDs, engineered mutants, and ligand complexes, which already includes over a dozen published crystal structures. The proposed studies address our major hypotheses that: ligand recognition and binding by collectins is combinatorial and driven by specific structural principles of complementarity and involves a cohort of specificity pockets and clefts, primarily on the uppermost surface of the carbohydrate recognition domain; and that these features differ in SP-A and SP-D, allowing them to each recognize a distinct lipid pool. This diversity increases the range of collectin-sensitive pathogens that can be recognized in human lung surfactant. The accumulated information from the proposed studies will provide a basis for future efforts to engineer more effective or more specific forms of collectin NCRD that may be used therapeutically in re.t;nnn.;e to immunnlnaical challenge from inhaled nathnaen.*; RELEVANCE (See instructions): The pulmonary collectins at the front line of defense against inhaled pathogens act as innate host defense molecules that can be used against inhaled bioterror agents, highly virulent new or emerging microbial strains, and antibiotic resistant pathogens. Mechanistic information can be used in designing novel therapeutics based on aerosolized forms of these proteins.